The present invention relates to a class of substituted imidazo-triazine derivatives and to their use in therapy. More particularly, this invention is concerned with imidazo[1,2-b][1,2,4]triazine analogues which are substituted in the 7-position by a substituted phenyl ring. These compounds are ligands for GABAA receptors and are therefore useful in the therapy of deleterious mental states.
Receptors for the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), are divided into two main classes: (1) GABAA receptors, which are members of the ligand-gated ion channel superfamily; and (2) GABAB receptors, which may be members of the G-protein linked receptor superfamily. Since the first cDNAs encoding individual GABAA receptor subunits were cloned the number of known members of the mammalian family has grown to include at least six xcex1 subunits, four xcex2 subunits, three xcex3 subunits, one xcex4 subunit, one xcex5 subunit and two xcfx81 subunits.
Although knowledge of the diversity of the GABAA receptor gene family represents a huge step forward in our understanding of this ligand-gated ion channel, insight into the extent of subtype diversity is still at an early stage. It has been indicated that an xcex1 subunit, a xcex2 subunit and a xcex3 subunit constitute the minimum requirement for forming a fully functional GABAA receptor expressed by transiently transfecting cDNAs into cells. As indicated above, xcex4, xcex5 and xcfx81 subunits also exist, but are present only to a minor extent in GABAA receptor populations.
Studies of receptor size and visualisation by electron microscopy conclude that, like other members of the ligand-gated ion channel family, the native GABAA receptor exists in pentameric form. The selection of at least one xcex1, one xcex2 and one xcex3 subunit from a repertoire of seventeen allows for the possible existence of more than 10,000 pentameric subunit combinations. Moreover, this calculation overlooks the additional permutations that would be possible if the arrangement of subunits around the ion channel had no constraints (i.e. there could be 120 possible variants for a receptor composed of five different subunits).
Receptor subtype assemblies which do exist include, amongst many others, xcex11xcex22xcex32, xcex12xcex2xcex31, xcex12xcex22/3xcex32, xcex13xcex2xcex32/3, xcex14xcex2xcex4, xcex15xcex23xcex32/3, xcex16xcex2xcex32 and xcex16xcex2xcex4. Subtype assemblies containing an xcex11 subunit are present in most areas of the brain and are thought to account for over 40% of GABAA receptors in the rat. Subtype assemblies containing xcex12 and xcex13 subunits respectively are thought to account for about 25% and 17% of GABAA receptors in the rat. Subtype assemblies containing an xcex15 subunit are expressed predominantly in the hippocampus and cortex and are thought to represent about 4% of GABAA receptors in the rat.
A characteristic property of all known GABAA receptors is the presence of a number of modulatory sites, one of which is the benzodiazepine (BZ) binding site. The BZ binding site is the most explored of the GABAA receptor modulatory sites, and is the site through which anxiolytic drugs such as diazepam and temazepam exert their effect. Before the cloning of the GABAA receptor gene family, the benzodiazepine binding site was historically subdivided into two subtypes, BZ1 and BZ2, on the basis of radioligand binding studies. The BZ1 subtype has been shown to be pharmacologically equivalent to a GABAA receptor comprising the xcex11 subunit in combination with a xcex2 subunit and xcex32. This is the most abundant GABAA receptor subtype, and is believed to represent almost half of all GABAA receptors in the brain.
Two other major populations are the xcex12xcex2xcex32 and xcex13xcex2xcex32/3 subtypes. Together these constitute approximately a further 35% of the total GABAA receptor repertoire. Pharmacologically this combination appears to be equivalent to the BZ2 subtype as defined previously by radioligand binding, although the BZ2 subtype may also include certain xcex15-containing subtype assemblies. The physiological role of these subtypes has hitherto been unclear because no sufficiently selective agonists or antagonists were known.
It is now believed that agents acting as BZ agonists at xcex11xcex2xcex32, xcex12xcex2xcex32 or xcex13xcex2xcex32 subtypes will possess desirable anxiolytic properties. Compounds which are modulators of the benzodiazepine binding site of the GABAA receptor by acting as BZ agonists are referred to hereinafter as xe2x80x9cGABAA receptor agonistsxe2x80x9d. The xcex11-selective GABAA receptor agonists alpidem and zolpidem are clinically prescribed as hypnotic agents, suggesting that at least some of the sedation associated with known anxiolytic drugs which act at the BZ1 binding site is mediated through GABAA receptors containing the xcex11 subunit. Accordingly, it is considered that GABAA receptor agonists which interact more favourably with the xcex12 and/or xcex13 subunit than with xcex11 will be effective in the treatment of anxiety with a reduced propensity to cause sedation. Also, agents which are antagonists or inverse agonists at xcex11 might be employed to reverse sedation or hypnosis caused by xcex11 agonists.
The compounds of the present invention, being selective ligands for GABAA receptors, are therefore of use in the treatment and/or prevention of a variety of disorders of the central nervous system. Such disorders include anxiety disorders, such as panic disorder with or without agoraphobia, agoraphobia without history of panic disorder, animal and other phobias including social phobias, obsessive-compulsive disorder, stress disorders including post-traumatic and acute stress disorder, and generalized or substance-induced anxiety disorder; neuroses; convulsions; migraine; depressive or bipolar disorders, for example single-episode or recurrent major depressive disorder, dysthymic disorder, bipolar I and bipolar II manic disorders, and cyclothymic disorder; psychotic disorders including schizophrenia; neurodegeneration arising from cerebral ischemia; attention deficit hyperactivity disorder; speech disorders, including stuttering; and disorders of circadian rhythm, e.g. in subjects suffering from the effects of jet lag or shift work.
Further disorders for which selective ligands for GABAA receptors may be of benefit include pain and nociception; emesis, including acute, delayed and anticipatory emesis, in particular emesis induced by chemotherapy or radiation, as well as motion sickness, and post-operative nausea and vomiting; eating disorders including anorexia nervosa and bulimia nervosa; premenstrual syndrome; muscle spasm or spasticity, e.g. in paraplegic patients; hearing disorders, including tinnitus and age-related hearing impairment; urinary incontinence; and the effects of substance abuse and dependency, including alcohol withdrawal. Selective ligands for GABAA receptors may also be effective as pre-medication prior to anaesthesia or minor procedures such as endoscopy, including gastric endoscopy.
In addition, the compounds in accordance with the present invention may be useful as radioligands in assays for detecting compounds capable of binding to the human GABAA receptor.
The present invention provides a class of imidazo-triazine derivatives which possess desirable binding properties at various GABAA receptor subtypes. The compounds in accordance with the present invention have good affinity as ligands for the xcex12 and/or xcex13 subunit of the human GABAA receptor. The compounds of this invention may interact more favourably with the xcex12 and/or xcex13 subunit than with the xcex11 subunit. Desirably, the compounds of the invention will exhibit functional selectivity in terms of a selective efficacy for the xcex12 and/or xcex13 subunit relative to the xcex11 subunit.
The compounds of the present invention are GABAA receptor subtype ligands having a binding affinity (Ki) for the xcex12 and/or xcex13 subunit, as measured in the assay described hereinbelow, of 200 nM or less, typically of 100 nM or less, and ideally of 20 nM or less. The compounds in accordance with this invention may possess at least a 2-fold, suitably at least a 5-fold, and advantageously at least a 10-fold, selective affinity for the xcex12 and/or xcex13 subunit relative to the xcex11 subunit. However, compounds which are not selective in terms of their binding affinity for the xcex12 and/or xcex13 subunit relative to the xcex11 subunit are also encompassed within the scope of the present invention; such compounds will desirably exhibit functional selectivity in terms of zero or weak (positive or negative) efficacy at the xcex11 subunit and a full or partial agonist profile at the xcex12 and/or xcex13 subunit.
The present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof: 
wherein
X1 represents hydrogen or fluoro;
R11 represents hydrogen, C1-6 alkyl, halo(C1-6)alkyl, dihalo(C1-6)alkyl, hydroxy(C1-6)alkyl, heteroaryl, halogen, trifluoromethyl, C1-6 alkoxy, formyl, C2-6 alkylcarbonyl, C2-6 alkoxycarbonyl or xe2x80x94CR4xe2x95x90NOR5;
R4 represents hydrogen or C1-6 alkyl;
R5 represents hydrogen, C1-6 alkyl, hydroxy(C1-6)alkyl or di(C1-6)alkylamino(C1-6)alkyl; and
R6 represents fluoro.
For use in medicine, the salts of the compounds of formula I will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
Suitable alkyl groups include straight-chained and branched alkyl groups containing from 1 to 6 carbon atoms. Typical examples include methyl and ethyl groups, and straight-chained or branched propyl, butyl and pentyl groups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl, isobutyl, tert-butyl and 2,2-dimethylpropyl. Derived expressions such as xe2x80x9cC1-6 alkoxyxe2x80x9d are to be construed accordingly.
Suitable heteroaryl groups include pyridinyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, benzofuryl, dibenzofuryl, thienyl, benzthienyl, pyrrolyl, indolyl, pyrazolyl, indazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, benzimidazolyl, oxadiazolyl, thiadiazolyl, triazolyl and tetrazolyl groups.
The term xe2x80x9chalogenxe2x80x9d as used herein includes fluorine, chlorine, bromine and iodine, especially fluoro or chloro.
Where the compounds according to the invention have at least one asymmetric centre, they may accordingly exist as enantiomers. Where the compounds according to the invention possess two or more asymmetric centres, they may additionally exist as diastereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.
In one embodiment, X1 represents hydrogen. In another embodiment, X1 represents fluoro.
Suitably, R4 represents hydrogen or methyl.
Suitably, R5 represents hydrogen, methyl, ethyl, hydroxyethyl or dimethylaminoethyl. Particular values of R5 include hydrogen, hydroxyethyl and dimethylaminoethyl.
Where R11 represents heteroaryl, this group is suitably furyl.
Illustrative values of R11 include hydrogen, methyl, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, difluoroethyl, hydroxyethyl, fluoropropyl, hydroxypropyl, tert-butyl, furyl, chloro, methoxy, formyl, acetyl, methoxycarbonyl and xe2x80x94CR2xe2x95x90NOR3, in which R2 and R3 are as defined above.
Specific values of R11 include hydrogen, methyl, difluoroethyl (especially 1,1-difluoroethyl), fluoropropyl (especially 2-fluoroprop-2-yl), hydroxypropyl (especially 2-hydroxyprop-2-yl), tert-butyl and trifluoromethyl.
In one embodiment, R11 represents methyl. In another embodiment, R11 represents trifluoromethyl. In a further embodiment, R11 represents 2-hydroxyprop-2-yl. In an additional embodiment, R11 represents 2-fluoroprop-2-yl.
The fluorine atom R6 is favourably attached to the phenyl ring at the 4-, 5- or 6-position (relative to the cyano group at position 2), preferably at the 6-position.
Specific compounds within the scope of the present invention include:
4,2xe2x80x2-difluoro-5xe2x80x2-[3-(1-fluoro-1-methylethyl)imidazo[1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile;
5,2xe2x80x2-difluoro-5xe2x80x2-(3-trifluoromethylimidazo[1,2-b][1,2,4]triazin-7-yl)-biphenyl-2-carbonitrile;
4,2xe2x80x2-difluoro-5xe2x80x2-[3-(1-hydroxy-1-methylethyl)imidazo [1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile;
4-fluoro-3xe2x80x2-[3-(1-hydroxy-1-methylethyl)imidazo [1,2-b][1,2,4]triazin-7-yl]-biphenyl-2-carbonitrile;
6,2xe2x80x2-difluoro-5xe2x80x2-[3-(1-hydroxy-1-methylethyl)imidazo [1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile;
and pharmaceutically acceptable salts thereof.
Also provided by the present invention is a method for the treatment and/or prevention of anxiety which comprises administering to a patient in need of such treatment an effective amount of a compound of formula I as defined above or a pharmaceutically acceptable salt thereof.
Further provided by the present invention is a method for the treatment and/or prevention of convulsions (e.g. in a patient suffering from epilepsy or a related disorder) which comprises administering to a patient in need of such treatment an effective amount of a compound of formula I as defined above or a pharmaceutically acceptable salt thereof.
The binding affinity (Ki) of the compounds according to the present invention for the xcex13 subunit of the human GABAA receptor is conveniently as measured in the assay described hereinbelow. The xcex13 subunit binding affinity (Ki) of the compounds of the invention is ideally 50 nM or less, preferably 10 nM or less, and more preferably 5 nM or less.
The compounds according to the present invention will ideally elicit at least a 40%, preferably at least a 50%, and more preferably at least a 60%, potentiation of the GABA EC20 response in stably transfected recombinant cell lines expressing the xcex13 subunit of the human GABAA receptor. Moreover, the compounds of the invention will ideally elicit at most a 30%, preferably at most a 20%, and more preferably at most a 10%, potentiation of the GABA EC20 response in stably transfected recombinant cell lines expressing the xcex11 subunit of the human GABAA receptor.
The potentiation of the GABA EC20 response in stably transfected cell lines expressing the xcex13 and xcex11 subunits of the human GABAA receptor can conveniently be measured by procedures analogous to the protocol described in Wafford et al., Mol. Pharmacol., 1996, 50, 670-678. The procedure will suitably be carried out utilising cultures of stably transfected eukaryotic cells, typically of stably transfected mouse Ltkxe2x88x92 fibroblast cells.
The compounds according to the present invention exhibit anxiolytic activity, as may be demonstrated by a positive response in the elevated plus maze and conditioned suppression of drinking-tests (cf Dawson et al., Psychopharmacology, 1995, 121, 109-117). Moreover, the compounds of the invention are substantially non-sedating, as may be confirmed by an appropriate result obtained from the response sensitivity (chain-pulling) test (cf. Bayley et al., J. Psychopharmacol., 1996, 10, 206-213).
The compounds according to the present invention may also exhibit anticonvulsant activity. This can be demonstrated by the ability to block pentylenetetrazole-induced seizures in rats and mice, following a protocol analogous to that described by Bristow et al. in J. Pharmacol. Exp. Ther., 1996, 279, 492-501.
In order to elicit their behavioural effects, the compounds of the invention will ideally be brain-penetrant; in other words, these compounds will be capable of crossing the so-called xe2x80x9cblood-brain barrierxe2x80x9d. Preferably, the compounds of the invention will be capable of exerting their beneficial therapeutic action following administration by the oral route.
The invention also provides pharmaceutical compositions comprising one or more compounds of this invention in association with a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums; and other. pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
In the treatment of anxiety, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, and especially about 0.05 to 5 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day.
The compounds in accordance with the present invention may be prepared by a process which comprises reacting a compound of formula II with a compound of formula III: 
wherein X1, R11 and R6 are as defined above, L1 represents a suitable leaving group, and M1 represents a boronic acid moiety xe2x80x94B(OH)2 or a cyclic ester thereof formed with an organic diol, e.g. pinacol or neopentyl glycol, or M1 represents xe2x80x94Sn(Alk)3 in which Alk represents a C1-6 alkyl group, typically n-butyl; in the presence of a transition metal catalyst.
The leaving group L1 is typically a halogen atom, e.g. bromo.
The transition metal catalyst of use in the reaction between compounds II and III is suitably tetrakis(triphenylphosphine)-palladium(0). The reaction is conveniently carried out at an elevated temperature in a solvent such as N,N-dimethylacetamide, 1,2-dimethoxyethane, tetrahydrofuran or 1,4-dioxane, advantageously in the presence of potassium phosphate, sodium carbonate or copper(I) iodide. Alternatively, the transition metal catalyst employed may be dichloro[1,1xe2x80x2-bis(diphenylphosphino)ferrocene]palladium(II), in which case the reaction may conveniently be carried out at an elevated temperature in a solvent such as N,N-dimethylformamide, typically in the presence of potassium phosphate.
In an alternative procedure, the compounds according to the present invention may be prepared by a process which comprises reacting a compound of formula IV with a compound of formula V: 
wherein X1, R11, R6, L1 and M1 are as defined above; in the presence of a transition metal catalyst; under conditions analogous to those described above for the reaction between compounds II and III.
Where M1 in the intermediates of formula III and IV above represents a boronic acid moiety xe2x80x94B(OH)2 or a cyclic ester thereof formed with pinacol or neopentyl glycol, the relevant compound III or IV may be prepared by reacting bis(pinacolato)diboron or bis(neopentyl glycolato)diborane respectively with a compound of formula VI or VII: 
wherein X1, R11 and R6 are as defined above, and L2 represents hydroxy or a suitable leaving group; in the presence of a transition metal catalyst.
Where L2 represents a leaving group, this is typically trifluoromethanesulfonyloxy (triflyloxy); or a halogen atom such as bromo.
The transition metal catalyst of use in the reaction between bis(pinacolato)diboron or bis(neopentyl glycolato)diborane and compound VI or VII is suitably dichloro[1,1xe2x80x2-bis(diphenylphosphino)ferrocene]-palladium(II). The reaction is conveniently carried out at an elevated temperature in a solvent such as 1,4-dioxane, optionally in admixture with dimethylsulfoxide, typically in the presence of 1,1xe2x80x2-bis(diphenylphosphino)ferrocene and/or potassium acetate.
Where L2 in the intermediates of formula VII above represents triflyloxy, the relevant compound may be prepared by reacting the corresponding compound of formula VII wherein L2 is hydroxy with N-phenyltriflylimide, typically in the presence of triethylamine; or with triflic anhydride, typically in the presence of pyridine. Analogous conditions may be utilised for converting an intermediate of formula VI above wherein L2 represents hydroxy into the corresponding compound wherein L2 represents triflyloxy.
The intermediates of formula VII above wherein L2 is hydroxy may suitably be prepared from the appropriate methoxy-substituted precursor of formula VIII: 
wherein X1 and R11 are as defined above; by treatment with boron tribromide, typically in chloroform; or with hydrogen bromide, typically in acetic acid at reflux.
The intermediates of formula VIII above may be prepared by reacting a compound of formula II as defined above with the appropriate compound of formula IX: 
wherein X1 and M1 are as defined above; in the presence of a transition metal catalyst; under conditions analogous to those described above for the reaction between compounds II and III.
Where L1 in the intermediates of formula II above represents bromo, this compound may be prepared by bromination of the corresponding compound of formula X: 
wherein R11 is as defined above; typically by treatment with bromine in acetic acid, in the presence of sodium acetate and optionally also potassium bromide.
The intermediates of formula X may be prepared by reacting bromoacetaldehyde with the requisite compound of formula XI: 
wherein R11 is as defined above.
The reaction is conveniently carried out by heating the reactants in 1,2-dimethoxyethane, or a lower alkanol such as methanol and/or ethanol, at a temperature typically in the region of 60-80xc2x0 C.
In a still further procedure, the compounds according to the present invention may be prepared by a process which comprises reacting a compound of formula XI as defined above with a compound of formula XII: 
wherein X1 and R6 are as defined above, and L3 represents a suitable leaving group; under conditions analogous to those described above for the reaction between bromoacetaldehyde and compound XI.
The leaving group L3 is suitably a halogen atom, e.g. bromo.
In a yet further procedure, the compounds according to the present invention wherein X1 represents hydrogen and R11 represents a heteroaryl moiety may be prepared by a process which comprises reacting a compound of formula XIII with a compound of formula XIV: 
wherein R6 is as defined above, R1a represents a heteroaryl moiety, and L4 represents a suitable leaving group; in the presence of a transition metal catalyst.
The leaving group L4 is typically a halogen atom, e.g. chloro.
The transition metal catalyst of use in the reaction between compounds XIII and XIV is suitably tris(dibenzylideneacetone)-dipalladium(0), in which case the reaction is conveniently effected at an elevated temperature in a solvent such as 1,4-dioxane, typically in the presence of tri-tert-butylphosphine and cesium carbonate.
Where L4 in the compounds of formula XIV above represents a halogen atom, these compounds correspond to compounds of formula I as defined above wherein R11 represents halogen, and they may therefore be prepared by any of the methods described above for the preparation of the compounds according to the invention.
Where they are not commercially available, the starting materials of formula V, IX, XI and XII may be prepared by methods analogous to those described in the accompanying Examples, or by standard methods well known from the art.
It will be understood that any compound of formula I initially obtained from any of the above processes may, where appropriate, subsequently be elaborated into a further compound of formula I by techniques known from the art. For example, a compound of formula I wherein R11 represents C2-6 alkoxycarbonyl initially obtained may be reduced with lithium aluminium hydride to the corresponding compound of formula I wherein R11 represents hydroxymethyl. The latter compound may then be oxidised to the corresponding compound of formula I wherein R11 represents formyl by treatment with manganese dioxide. The formyl derivative thereby obtained may be condensed with a hydroxylamine derivative of formula H2Nxe2x80x94OR5 to provide a compound of formula I wherein R11 represents xe2x80x94CHxe2x95x90NOR5. Alternatively, the compound of formula I wherein R11 represents formyl may be reacted with a Grignard reagent of formula RaMgBr, wherein Ra represents C1-5 alkyl, to afford a compound of formula I wherein R11 represents xe2x80x94CH(OH)Ra, and this compound may in turn be oxidised using manganese dioxide to the corresponding compound of formula I wherein R11 represents xe2x80x94CORa. The latter compound may then be condensed with a hydroxylamine derivative of formula H2Nxe2x80x94OR5 to provide a compound of formula I wherein R11 represents xe2x80x94CRaxe2x95x90NOR5.
Where a mixture of products is obtained from any of the processes described above for the preparation of compounds according to the invention, the desired product can be separated therefrom at an appropriate stage by conventional methods such as preparative HPLC; or column chromatography utilising, for example, silica and/or alumina in conjunction with an appropriate solvent system.
Where the above-described processes for the preparation of the compounds according to the invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The novel compounds may be prepared in racemic form,. or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The novel compounds may, for example, be resolved into their component enantiomers by standard techniques such as preparative HPLC, or the formation of diastereomeric pairs by salt formation with an optically active acid, such as (xe2x88x92)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-1-tartaric acid, followed by fractional crystallization and regeneration of the free base. The novel compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The following Examples illustrate the preparation of compounds according to the invention.
The compounds in accordance with this invention potently inhibit the binding of [3H]-flumazenil to the benzodiazepine binding site of human GABAA receptors containing the xcex12 and/or xcex13 subunit stably expressed in Ltkxe2x88x92 cells.
Reagents
Phosphate buffered saline (PBS).
Assay buffer: 10 mM KH2PO4, 100 mM KCl, pH 7.4 at room temperature.
[3H]-Flumazenil (18 nM for xcex11xcex23xcex32 cells; 18 nM for xcex12xcex23xcex32 cells; 10 nM for xcex13xcex23xcex32 cells) in assay buffer.
Flunitrazepam 100 xcexcM in assay buffer.
Cells resuspended in assay buffer (1 tray to 10 ml).
Harvesting Cells
Supernatant is removed from cells. PBS (approximately 20 ml) is added. The cells are scraped and placed in a 50 ml centrifuge tube. The procedure is repeated with a further 10 ml of PBS to ensure that most of the cells are removed. The cells are pelleted by centrifuging for 20 min at 3000 rpm in a benchtop centrifuge, and then frozen if desired. The pellets are resuspended in 10 ml of buffer per tray (25 cmxc3x9725 cm) of cells.
Assay
Can be carried out in deep 96-well plates or in tubes. Each tube contains:
300 xcexcl of assay buffer.
50 xcexcl of [3H]-flumazenil (final concentration for xcex11xcex23xcex32: 1.8 nM; for xcex12xcex23xcex32: 1.8 nM; for xcex13xcex23xcex32: 1.0 nM).
50 xcexcl of buffer or solvent carrier (e.g. 10% DMSO) if compounds are dissolved in 10% DMSO (total); test compound or flunitrazepam (to determine non-specific binding), 10 xcexcM final concentration.
100 xcexcl of cells.
Assays are incubated for 1 hour at 40xc2x0 C., then filtered using either a Tomtec or Brandel cell harvester onto GF/B filters followed by 3xc3x973 ml washes with ice cold assay buffer. Filters are dried and counted by liquid scintillation counting. Expected values for total binding are 3000-4000 dpm for total counts and less than 200 dpm for non-specific binding if using liquid scintillation counting, or 1500-2000 dpm for total counts and less than 200 dpm for non-specific binding if counting with meltilex solid scintillant. Binding parameters are determined by non-linear least squares regression analysis, from which the inhibition constant Ki can be calculated for each test compound.
The compounds of the accompanying Examples were tested in the above assay, and all were found to possess a Ki value for displacement of [3H]-flumazenil from the xcex12 and/or xcex13 subunit of the human GABAA receptor of 100 nM or less.
7-Bromo-3-trifluoromethylimidazo[1,2-b][1,2,4]triazine
a) 3-Amino-5-trifluoromethyl-1,2.4-triazine
To a stirred solution of sodium acetate trihydrate (22.62 g, 166.2 mmol) in water (80 ml) was added 1,1-dibromo-3,3,3-trifluoroacetone (21.57 g, 79.9 mmol). The solution was heated at reflux under nitrogen for 30 min, then allowed to cool to room temperature before adding solid aminoguanidine bicarbonate (10.88 g, 79.9 mmol). The resulting pale yellow solution (pH 5) was stirred at room temperature for 3 h, then 4 N aqueous NaOH solution (40 ml, 160 mmol) was added causing a precipitate to appear. The mixture (pH 10) was stirred under nitrogen for a further 39 h. The solid was collected by filtration, washed with water and dried at 60xc2x0 C. under vacuum to give 6.96 g of a mixture of two isomers in a 28:72 ratio. This was further purified by flash chromatography (silica gel, 30% EtOAc/isohexane), then recrystallised from ethanol to afford 3.53 g (27%) of the title compound: 1H NMR (400 MHz, DMSO-d6) xcex48.00 (2H, br s), 9.08 (1H, s).
b) 3-Trifluoromethylimidazo[1,2-b][1,2,4]triazine
A stirred mixture of bromoacetaldehyde diethyl acetal (2.30 ml, 14.8 mmol) in concentrated hydrobromic acid (0.73 ml) and water (0.73 ml) was heated at reflux for 2 h, then poured into ethanol (25 ml). The solution was neutralised to pH 7 with solid sodium hydrogencarbonate, then filtered. To the filtrate was added 3-amino-5-trifluoromethyl-1,2,4-triazine (1.0079 g, 6.14 mmol) and the mixture was stirred at 60xc2x0 C. for 20 h, then 80xc2x0 C. for 23 h. The mixture was evaporated in vacuo, and the residue was purified by flash chromatography (silica gel, 35-50% EtOAc/isohexane) to give 0.2593 g (22%) of the title compound: 1H NMR (360 MHz, CDCl3) xcex48.20 (1H, d, J 0.8 Hz), 8.30 (1H, d, J 0.9 Hz), 8.73 (1H, s).
c) 7-Bromo-3-trifluoromethylimidazo[1,2-b][1,2,4]triazine
To a solution of 3-trifluoromethylimidazo[1,2-b][1,2,4]triazine (0.2211 g, 1.18 mmol) in acetic acid (6 ml) was added sodium acetate (0.1470 g, 1.79 mmol), then bromine (90.8 xcexcl, 1.76 mmol). The solution was stirred at room temperature for 6 h, then partitioned between saturated aqueous NaHCO3 (100 ml) and ethyl acetate (100 ml). The aqueous layer (pH 9) was further extracted with ethyl acetate (100 ml), and the combined organic extracts were dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 25% EtOAc/isohexane) to afford 0.2073 g (66%) of the title compound: 1H NMR (360 MHz, CDCl3) xcex48.30 (1H, s), 8.83 (1H, s).
2-(2-Fluoro-5-nitrophenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane
A mixture of 2-bromo-1-fluoro-4-nitrobenzene (A. Groweiss, Org. Process Res. Dev., 2000, 4, 30-33) (50.10 g, 0.228 mol), dried potassium acetate (44.70 g, 0.455 mol) and bis(pinacolato)diboron (59.16 g, 0.233 mol) in 1,4-dioxane (539 ml) and dimethylsulfoxide (11 ml) was degassed by bubbling nitrogen through the mixture for 1 h. Dichloro[1,1xe2x80x2-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (5.58 g, 6.83 mmol) was added and the mixture was stirred at 90xc2x0 C. under nitrogen for 18.5 h, adding more bis(pinacolato)diboron (7.34 g, 0.029 mol) after 2.5 h. After allowing to cool, the mixture was filtered through glass fibre paper, and the solid was washed with a little dichloromethane. The combined filtrates were evaporated in vacuo and the residue was partitioned between 2 M aqueous NaOH (800 ml) and diethyl ether (800 ml). The aqueous layer was then acidified to pH 6 with concentrated hydrochloric acid (120 ml), causing a solid to precipitate. After leaving in a fridge for 3 days, the solid was collected by filtration, washed with water and dried under vacuum to leave 54.82 g (90%) of 2-(2-fluoro-5-nitrophenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane: 1H NMR (360 MHz, DMSO-d6) xcex41.33 (12H, s), 7.48 (1H, m), 8.40-8.45 (2H, m).
3-(1-Fluoro-1-methylethyl)-7-[4-fluoro-3-(pyridin-3-yl)phenyl]imidazo[1,2-b][1,2,4]triazine, dihydrochloride salt
a) 3-Methyl-3-fluoro-2-butanone
This was prepared from 3-bromo-3-methyl-2-butanone as described by Fry and Migron (Tetrahedron Lett., 1979, 3357-3360) to give, after distillation using a Vigreux column, a 47% yield of a 94:6 mixture of the title compound and 3-methyl-3-buten-2-one as a colourless oil: bp 74-6xc2x0 C.; 1H NMR (360 MHz, CDCl3) xcex41.45 (6H, d, J 21.5 Hz), 2.28 (3H, d, J 5.0 Hz).
b) 1,1-Dibromo-3-fluoro-3-methyl-2-butanone
To a stirred solution of 3-methyl-3-fluoro-2-butanone (0.1031 g, 0.990 mmol) in anhydrous dichloromethane (5 ml) under nitrogen was added solid pyridinium tribromide (0.7035 g, 1.98 mmol) and the mixture was stirred at room temperature for 18 h. The mixture was then diluted with dichloromethane (5 ml), washed with dilute aqueous sodium hydrogensulfite (10 ml), then saturated aqueous NaCl (10 ml), dried (Na2SO4) and evaporated under low vacuum with no heat. The residue was purified by flash chromatography [silica gel, 5% Et2O/petroleum ether (40-60xc2x0 C.)] to afford 0.1869 g (72%) of the title compound as a colourless oil: 1H NMR (360 MHz, CDCl3) xcex41.65 (6H, d, J 21.5 Hz), 6.51 (1H, d, J 1.5 Hz).
c) 3-Amino-5-(1-fluoro-1-methylethyl)-1,2,4-triazine
This was prepared in 45% yield as a single isomer by a similar procedure to that described in Example A, step a, except using 1,1-dibromo-3-fluoro-3-methyl-2-butanone instead of 1,1-dibromo-3,3,3-trifluoroacetone: 1H NMR (360 MHz, DMSO-d6) xcex41.63 (6H, d, J 8.0 Hz), 7.32 (2H, br s), 8.73 (1H, d, J 1.0 Hz); MS (ES+) m/z 157 [M+H]+.
d) 3-(1-Fluoro-1-methylethyl)imidazo[1,2-b][1,2,4]triazine
A stirred mixture of bromoacetaldehyde diethyl acetal (1.20 ml, 7.73 mmol) in concentrated hydrobromic acid (0.38 ml) and water (0.38 ml) was heated at reflux for 40 min, then poured into ethanol (3 ml). The solution was neutralised to pH 7 with solid sodium hydrogencarbonate, then filtered, washing the solid with more ethanol (3 ml). To the filtrate was added 3-amino-5-(1-fluoro-1-methylethyl)-1,2,4-triazine (1.0046 g, 6.43 mmol) and the mixture was stirred at 70-80xc2x0 C. for 17 h. The mixture was evaporated in vacuo, and the residue was purified by flash chromatography (silica gel, 70% EtOAc/isohexane to 15% MeOH/EtOAc, then 20% EtOAc/CH2Cl2) to give 0.2000 g (17%) of the title compound as a pale yellow solid: 1H NMR (360 MHz, CDCl3) xcex41.82 (6H, d, J 22.1 Hz), 7.97 (1H, d, J 1.3 Hz), 7.99 (1H, d, J 1.2 Hz), 8.69 (1H, d, J 1.0 Hz).
e) 7-Bromo-3-(1-fluoro-1-methylethyl)imidazo[1,2-b][1,2,4]triazine
This was prepared in 92% yield by a similar procedure to that described in Example A, step c, except using 3-(1-fluoro-1-methylethyl)imidazo[1,2-b][1,2,4]triazine instead of 3-trifluoromethylimidazo[1,2-b][1,2,4]triazine: 1H NMR (360 MHz, CDCl3) xcex41.82 (6H, d, J 22.1 Hz), 7.99 (1H, s), 8.81 (1H, d, J 1.1 Hz).
f) 3-(1-Fluoro-1-methylethyl)-7-[4-fluoro-3-(pyridin-3-yl)phenyl]imidazo[1,2-b][1,2,4]triazine, dihydrochloride salt 
To a solution of 2-bromo-1-fluoro-4-nitrobenzene (A. Groweiss, Org. Process Res. Dev., 2000, 4, 30-33) in tetrahydrofuran (75 ml) and ethanol (75 ml) was added tin(II) chloride dihydrate and the mixture left to stir at ambient temperature for 4 h. The solvent was evaporated and the residue was treated with ice-cold 2 N sodium hydroxide solution (200 ml). The resulting slurry was stirred for 30 min then extracted with dichloromethane (3xc3x97200 ml). The combined organic phase was washed with water (200 ml) and brine (200 ml), dried (MgSO4), filtered and evaporated to give 3-bromo-4-fluorophenylamine (7.92 g, 92%) as a yellow oil: 1H NMR (360 MHz, CDCl3) xcex43.53 (2H, s), 6.53-6.57 (1H, m), 6.83-6.85 (1H, m), 6.90 (1H, dd, J 9, 9 Hz).
A mixture of 3-bromo-4-fluorophenylamine (7.92 g, 41.7 mmol), diethyl(3-pyridyl)borane (6.74 g, 45.9 mmol), tetrakis(triphenylphosphine)palladium(0) (0.96 g, 0.83 mmol) and potassium carbonate (17.26 g, 125 mmol) in 1,2-dimethoxyethane (30 ml) and water (15 ml) was heated at 80xc2x0 C. for 20 h. After cooling to ambient temperature the reaction was partitioned between ethyl acetate (500 ml) and water (500 ml). The organics were washed with brine (400 ml), dried (Na2SO4), filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica gel, 0%-20% EtOAc/CH2Cl2) gave 4-fluoro-3-(pyridin-3-yl)phenylamine (3.64 g, 46%) as a colourless oil that solidified on standing to afford a white solid: 1H NMR (360 MHz, CDCl3) xcex43.65 (2H, s), 6.65-6.72 (2H, m), 6.99. (1H, dd, J 9, 9 Hz), 7.33-7.37 (1H, m), 7.84-7.86 (1H, m), 8.58 (1H, d, J 4 Hz), 8.76 (1H, m).
A warm solution of 4-fluoro-3-(pyridin-3-yl)phenylamine (3.64 g, 19.3 mmol) in 1,4-dioxane (10 ml) was treated with a solution of 48% aqueous hydrobromic acid (100 ml). The resulting suspension was cooled to 0xc2x0 C. before being treated dropwise over 20 min with a solution of sodium nitrite (1.53 g, 22.2 mmol) in water (4 ml). After stirring at 0xc2x0 C. for 2 h, a cooled (0xc2x0 C.) solution of copper(I) bromide (8.31 g, 57.9 mmol) in 48% aqueous hydrobromic acid (30 ml) was added to the reaction which was stirred at 0xc2x0 C. for 10 min then heated at 50xc2x0 C. for 20 min. The reaction was cooled to ambient temperature, poured onto ice-cold concentrated ammonia (500 ml) and the product was extracted into ethyl acetate (500 ml). The organics were washed with water (300 ml) and brine (300 ml), dried (Na2SO4), filtered and concentrated in vacuo to give a dark oil. Purification by dry flash column chromatography (silica gel, 10-30% EtOAc/isohexane) gave 3-(5-bromo-2-fluorophenyl)pyridine (3.1 g, 64%) as a white solid: 1H NMR (360 MHz, CDCl3) xcex47.09 (1H, dd, J 9, 1 Hz), 7.37-7.40 (1H, m), 7.46-7.51 (1H, m), 7.56-7.59 (1H, m), 7.83-7.86 (1H, m), 8.63-8.65 (1H, m), 8.77-8.79 (1H, m).
3-(5-Bromo-2-fluorophenyl)pyridine (3.1 g, 12.3 mmol), potassium acetate (3.62 g, 36.9 mmol) and bis(pinacolato)diboron (3.75 g, 14.8 mmol) were dissolved in 1,4-dioxane (40 ml) and dimethylsulfoxide (0.8 ml) and the mixture degassed with N2 for 15 min. Dichloro[1,1xe2x80x2-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (300 mg, 0.37 mmol) was added and the mixture heated at 90xc2x0 C. for 18 h. The mixture was cooled to ambient temperature-and partitioned between diethyl ether (200 ml) and 2 N hydrochloric acid (50 ml). The organics were discarded and the aqueous phase adjusted to pH 8 by the addition of 4 N sodium hydroxide solution and extracted with diethyl ether (2xc3x97500 ml). The organic layer was washed with brine (50 ml), dried (Na2SO4), filtered and pre-adsorbed onto silica. Purification by flash column chromatography (silica gel, 25% EtOAc/isohexane) gave 3-[2-fluoro-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)phenyl]pyridine (2.64 g, 72%) as a yellow oil that crystallised on standing: 1H NMR (36.0 MHz, CDCl3) xcex41.35 (12H, s), 7.20 (1H, dd, J 10, 8 Hz), 7.35-7.39 (1H, m), 7.81-7.91 (3H, m), 8.61 (1H, dd, J 5, 2 Hz), 8.82 (1H, s).
A stirred mixture of 7-bromo-3-(1-fluoro-1-methylethyl)imidazo[1,2-b][1,2,4]triazine (0.1059 g, 0.409 mmol) and 3-[2-fluoro-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)phenyl]pyridine (0.1832 g, 0.612 mmol) in 1,2-dimethoxyethane (2 ml) and 2 M aqueous Na2CO3 (0.613 ml, 1.23 mmol) was degassed by bubbling through nitrogen for 15 min. Tetrakis(triphenylphosphine)palladium(0) (23.4 mg, 0.020 mmol) was then added and the mixture was stirred at 80xc2x0 C. for 16 h under nitrogen. The mixture was partitioned between ethyl acetate (25 ml) and water (10 ml) and the aqueous phase was extracted further with ethyl acetate (2xc3x9725 ml). The organic extracts were combined, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, EtOAc) to yield 0.1047 g (73%) of the title compound as a yellow oil. The hydrochloride salt was prepared in diethyl ether: mp 113-126xc2x0 C.; 1H NMR (400 MHz, DMSO-d6) xcex41.81 (6H, d, J 22.2 Hz), 7.68 (1H, dd, J 10.6, 8.6 Hz), 8.03 (1H, dd, J 7.8, 5.5 Hz), 8.36 (1H, m), 8.43 (1H, dd, J 7.4, 2.3 Hz), 8.63 (1H, dd, J 7.4, 1.2 Hz), 8.67 (1H, s), 8.91 (1H, d, J 4.3 Hz), 9.07 (1H, d, J 0.8 Hz), 9.16 (1H, s); MS (ES+) m/z 352 [M+H]+. Anal. Found: C, 51.65; H, 4.48; N, 15.28%. Required for C19H15F2N5.2HCl.0.07C4H10O.H2O: C, 51.75; H, 4.44; N, 15.65%.